Chemotherapeutic compositions including esters of indolyl-3-acetic acid



United States Patent 3,326,766 CHEMOTHERAPEUTIC COMPOSITIONS IN- CLUDINGESTERS OF INDOLYL-3-ACETIC ACID David Norman, Redondo Beach, and RobertD. Schultz,

Whittier, Calif., assiguors to North American Aviation, Inc. No Drawing.Filed Mar. 20, 1964, Ser. No. 353,617 17 Claims- (Cl. 167-78) In thepractice of this invention according to a preferred embodiment there isprovided a treatment which effects a carcinolytic action comprisingexposing Ehrlich ascites tumor cells to the action of an ester of anauxin. Esters of auxins that are preferred include alkyl esters ofindo1yl-3-acetic acid (indoleacetic acid) and naphthyll-acetic acidwherein the alkyl group has from one to twelve carbon atoms. It has beenfound that aqueous pharmaceutical preparations of such esters areeffective in killing Ehrlich ascites tumor cells in tumor infected mice.

It appears that concentrations of greater than 2X10- molarindolyl-3-acetic acid in a tumor is toxic to the tumor cells in vitro.It is postulated that lower concentrations of indolyl-B-acetic acid arenormally present in cells as a natural intermediate in the metabolism ofthe cells. Treatment employing indolyl-3-acetic acid ornaphthyl-l-acetic acid as a carcinolytic agent is not effective becauseof the relative toxicity of these materials in normal tissue, and asomewhat protective effect which has been found when tumor cells areexposed to indolyl- 3-acetic acid or naphthyl-l-acetic acid.

Modification of the indolyl-3-acetic acid to the ethyl ester thereofshowed a pronounced carcinolytic action in vitro, however, the tumorcells rapidly degrade the ethyl ester, thereby reducing the efiicacy ofthis material as a chemotherapeutic agent. Further modification of' thechemical structure of the auxin in order to reduce the rate ofdegradation and the toxicity of the agents produces agents having acarcinolytic activity with minimized effect on normal tissue.

One group of auxin esters has the structural formula II CHz-C-O-R whereR is an alkyl group having from one to twelve carbon atoms substitutedfor the hydrogen of indolyl-3-acetic acid. Higher numbers of carbonatoms may yield chains 3,326,766 Patented June 20, 1967 "ice where R isan alkyl group having from one to twelve carbon atoms substituted forthe hydrogen of the napthyl l-acetic acid. 'I-Iigher numbers of carbonatoms yield esters that show increased toxicity in aqueous suspensionswithout increased eificacy. Significantly increased carcinolyticactivity has been found in the group of esters of napthyll-acetic acidwhen R is selected from the group consisting of methyl, ethyl, normalpropyl, and normal pentyl.

It has been found that the auxin esters possess a tolerated dosage levelthat is dependent on the alkyl group substituted on the auxin. Themaximum dosage that is safely tolerated without toxic side effects isindicated by reference to the LD set forth hereinafter. The LD of amaterial is the dosage level having lethal toxicity for about 50% of thetest animals to which the material is administered.

Generally, however, it has been found preferable to administer fromabout 200 to about 600 milligrams of auxin ester per kilogram of bodyweight of mice. Lower concentrations may not be completely effective inall cases and higher doses are seldom required to kill all freemalignant tumor cells.

Administration is preferably with the aid of a pharmaceutical carrier.The auxin esters are soluble in many oily materials commonly employed aspharmaceutical carriers such as, for example, mineral oil, lard,cottonseed oil, peanut oil, sesame oil or the like, and are insoluble inwater or in aqueous vehicles such as saline, glucose, gelatine or thelike. In vitro tests of the auxin esters suspended in Krebs-Ringerglucose solution established the carcinolytic activity of the auxinesters and in vivo tests were conducted for candidate materials havingtolerable LD values. However, it was found that administration of theauxin esters in solution in the tested oily pharmaceutical carriers gaveno measurable effect on tumor infected mice.

Unexpectedly it was found that similar concentrations in aqueoussuspension demonstrated a beneficial effect. The auxin ester issuspended in the aqueous medium by a suspending, emulsifying, or surfaceactive agent. Preferably a combination of surface active agent andemulsifier are employed in saline to prepare a suspension of the auxinester. Typical materials employed in the preparation of aqueoussuspensions include gum arabic', methyl cellulose, acetylatedmonoglyceride (available commercially as Myvacet from DistillationProducts Industry, a division of Eastman Kodak Company), monomethylglyceride, dimethyl glyceride or a moderately high molecular weightpolysorbitan (commercially available under the trade names Tween or Spanfrom Atlas Powder Company, Wilmington, Delaware). Other materialsemployed in the preparation of chemotherapeutic compositions containingan auxin ester may include glutathione, 1,2 propanediol, glycerol andglucose. Additionally the pH of the composition is adjusted by use of anaqueous solution such as tris(hydroxymethyl) aminomethane (tris buffer).

It is postulated that the oily pharmaceutical carriers which dissolvethe auxin esters and permit high dosages also serve as a preferentialsolvent so that little of the drug can enter the cell metabolic systembefore degradation. Therefore such carriers are ineffective and testsemploying such vehicles give an indication that the candidate drugs areineffective. It is found, however, that the auxin esters are effectivewhen appropriately administered, that is, in an aqueous vehicle. Asuspending agent or combination of suspending agents is employed todisperse the insoluble auxin ester and permit interaction with tumorcells. Likewise it has been found valuable to provide a water solublematerial in which the auxin ester is somewhat soluble in thepharmaceutical carrier. Thus,

for example, a material such as 1,2 propanediol is employed in thevehicle to provide a small solubility of the auxin ester in the vehicle.This probably permits more rapid entering of the auxin ester into themetabolic chain of the tumor cells.

It is preferred that the auxin ester be suspended in the aqueous mediumwith a concentration in the range of from two to 50 mg./ml. Lowerconcentrations than two mg./m1. require the administration of needlessquantities of liquid which must be assimilated by the mice. Higherconcentrations than 50 mg./ml. are difficult to maintain in suspensionand are preferably avoided.

It has been found in tests made in vivo that the alkyl esters of auxinscause a reversion of the Ehrlich tumor to a less virulent form. TheEhrlich carcinoma is a strain originally identified as a solid tumor. Inpropagating this tumor a liquid ascites tumor was isolated by Ehrlichand the resultant Ehrlich ascites carcinoma is a commonly usedtransplantable liquid tumor. The liquid ascites tumor is considerablymore virulent than the solid form of the Ehrlich carcinoma.

A conventional technique of propagating the liquid Ehrlich ascitescarcinoma for laboratory study was employed to maintain the strain. MaleWebster white Swiss mice infected with the tumor were sacrificed and theascitic fluid was collected. This fluid containing a high concentrationof malignant cells was diluted with physiological saline and an aliquotwas intraperitoneally injected into healthy mice to cause a malignantgrowth therein. Some of these mice having transplanted tumors were latersacrificed to provide ascitic fluid having malignant tumor cells tofurther propagate the tumor in additional animals. Some animals havingtransplanted liquid tumors were employed in examples set forthhereinafter.

In the course of tests, some of the animals infected with the liquidEhrlich ascites carcinoma received treatment with an auxin ester. It wasfound that treatment with the tertiary butyl ester of indolyl-3-aceticacid or with the normal pentyl ester of indolyl-3-acetic acid caused areversion of the liquid Ehrlich ascites carcinoma to a semi-solidEhrlich ascites carcinoma which formed in the peritoneal cavity on thesurfaces therein. The semi-solid tumor was collected from sacrificedmice, macerated and suspended in physiological saline. A portion of thissuspension was intraperitoneally injected in healthy mice to transplantthe resultant tumor. In each instance, it was found that the mice havingthe transplanted semi-solid tumor cells developed a semi-solid tumorhaving the same apparent characteristics as the parent semi-solid tumor.The semi-solid tumor produced by reversion after treatment with thenormal pentyl ester of indolyl-3-acetic acid was propagated in manyanimals to maintain the strain. Some of the animals having transplantedsemi-solid Ehrlich ascites carcinoma were employed in examples set forthhereinafter.

The treatment which caused the reversion of the liquid tumor to thesemi-solid tumor comprised a single one milliliter intraperitonealinjection of drug one day after transplanting the liquid tumor. One drugcomprised 25 mg./ml. normal pentyl ester of indolyl-3-acetic acid, 50mg./ml. glucose, 18 mg./ml. 1,2 pr-opanediol, 3 mg./ml. Myvacet, and 5mg./ ml. Tween 20 diluted to volume with 0.5% saline and buffered to 7.2to 7.4 with tris buffer. Another drug comprised mg./ml. of tertiarybutyl ester of ind0lyl-3-acetic acid and the same vehicle as recitedabove. In each instance a semi-solid tumor was found upon sacrifice ofthe animals. The tumor resulting from the normal pentyl ester treatmentwas employed in examples set forth hereinafter.

The semi-solid strain is a reversion of the liquid Ehrlich ascitescarcinoma toward the original solid tumor. The semi-solid reverted formof Ehrlich ascites carcinoma is less virulent than the liquid tumor fromwhich it originated and more virulent than the parent solid tumor. Forthe purposes of the examples set forth hereinafter, reference to asemi-solid Ehrlich ascites carcinoma concerns a strain propagated from atumor caused to revert by treatment with the normal pentyl ester ofindolyl-3-acetic acid.

The mechanism of action of the auxin esters in killing Ehrlich carcinomacells and in prolonging the life of infected mice is not specificallyknown. Because of the observed reversion of the liquid tumor to asemi-solid tumor, it is postulated that the auxin ester causes areversion of cells to a less virulent and weaker form even when no grossmorphological changes are observed in the tumor. This hypothesis isstrengthened by the observed reversion of transplanted semi-solid tumorsto solid tumors upon treatment comprising a single one milliliterintraperitoneal injection of drug comprising 20 mg./ml. normal pentylester of napthyl-l-acetic acid and 50 mg./ ml. glucose in the abovedescribed vehicle, or with similar treatment with the tertiary butylester of indolyl-3-acetic acid at a concentration of 20 mg./ml. andglucose at 50 mg./ml.

Analogy to the growth regulating properties of the indolyl-3-acetic acidand napthyl-l-acetic acid in plant tissue and the growth regulatingeffects of related compounds forms the basis for a theory whichattributes the carcinolytic activity to the location of active sites onthe molecule and the steric configuration of the molecule. The stericconfigurations of the indolyl-3-acetic acid and the esters thereof andthe esters of napthyl-l-acetic acid are sufficiently similar that if theester is present in suflicient concentration it may react in themetabolic chain of the cells to the exclusion of the indolyl-3-aceticacid thereby interrupting the metabolic chain and causing eventual deathof the tumor cells.

The following examples are given to more fully illustrate the principlesof this invention.

EXAMPLE 1 Free Ehrlich ascites tumor cells were collected from male,Webster White Swiss mice (initial body weight between 18 and 22 grams)at 6 to 10 days post transplantation, by drainage of the ascitic fluidfrom the abdominal cavity. One ml. of this fluid, which contained about3 10 free tumor cells per milliliter, was diluted with 10 ml.Krebs-Ringer phosphate solution, pH 6.8, containing glucose (1 mg./ml.).A 3 to 5-ml. aliquot of this intermediate mixture was diluted with anequal volume of the Krebs-Ringer phosphate-glucose solution, in whichthe relatively insoluble ethyl ester of indolyl-3-acetic acid (1mg./ml.) was suspended in gum arabic (1 mg./ml.). Each ascites tumor wasused as its own control, in that the tumor fluid was diluted to the samefinal cell suspension mixture, including gum arabic, but without ethylester of indolyl-3-acetic acid. Incubations were carried out inglass-stoppered, dark SO-ml. Ehrlenmeyer flasks for a maximum of sixhours at 36 C., or 20 hours at 22 0, without shaking. The cellsuspensions were sampled at various time intervals, and free tumor cellcounts were made in triplicate immediately following appropriatedilution in Tyrode-eosin solution. Total cell counts and stained cellcounts were made. The stained cells were considered to represent deadcells. The Tyrode-eosin stain employed comprises 16 gm. NaCl; 400 mg.KCl; 200' mg. CaCl 430 mg. MgCl -6H O; 230 mg. NaH PO -H O; 2 gm.glucose; 2 gm. NaHCO 1 gm. Eosin yellowish; and sufficient water to maketwo liters.

The results show a lethal effect of ethyl ester of indolyl- 3-aceticacid on the free ascites carcinoma cells-- percent mortality wasobserved at various times, all less than five hours, depending on thesensitivity of the tumor to this agentwhereas the controls exhibitedlittle or no death during the same period of incubation.

EXAMPLE 2 As in the above example, free ascites tumor cells werecollected from male, Webster white Swiss mice (initial body weightbetween 18 and 22 grams) at 6 to 10 days post transplantation bydraining the ascitic fluid from the abdominal cavity. On the average,the fluid contained about 3 10 cells/ml. In the present example, theascitic fluids from a number of different animals were pooled anddiluted by a factor of twenty with the Krebs-Ringer phosphate solution,pH 7.15. The final mixture contained glucose at 1 mg./ml. and thesubstantially insoluble auxin ester at 0.5 mg./ml. suspended with gumarabic at 1 mg./ml. Controls were prepared in an identical manner,except for the omission of the auxin esters. Incubation at 37 C. andcell counting of samples withdrawn at various intervals were performedas described above.

The Krebs-Ringer-glucose solution is a medium designed only forshort-term studies with isolated, live animal tissues, and the mediumcannot support the life of the tumor cells indefinitely. After a fourhour incubation at 37 C., some cell mortality could be observed incontrols of some tumor preparation; in other preparations, littlemortality Was observed even after siX hours. However, a 0.5 mg./ml.suspension of indoly1-3-acetic acid or of napthyl-l acetic acidappreciably reduced the rate of cell mortality of a sensitive tumorpreparation. In contrast, the ethyl ester of indlyl-3-acetic acid killed39% and the methyl ester of napthyl-l-acetic acid killed 23%, of suchtumor cells in two hours, compared to less than 4% death in theuntreated controls. After six hours, the auxin esters had killed 100% ofthe cells, compared to about a 25% mortality of the controls, and only a17% death of those cells treated with the aforementioned acids.

Similar tests were conducted with other alkyl esters of indolyl-3-aceticacid against the diluted Ehrlich ascites carcinoma in vitro. Table Iillustrates the results of these tests.

EXAMPLE 4 Life time tests were conducted on male Webster white Swissmice (18-22 grams body weight) to determine the efiicacy of the auxinester in vivo. Tumors were transplanted into said mice byintraperitoneal injection of 0.1 ml. of ascitic fluid collected from theother similar mice known to be infected with Ehrlich ascites carcinoma.The injection contained about 3X10 cells of Ehrlich ascites carcinoma.Drug treatment commenced the following day and involved a singleintraperitoneal injection of the auxin ester or injections of thevehicle or saline solution to serve as controls. The vehicle employedfor the in vivo tests comprised 1,2 propanediol, acetylatedmonoglyceride (Myvacet) and a polysorbitan having an average molecularWeight of about 20,000 (Tween 20) made up to the proper volume with 0.5%saline and buffered to pH 7.2- 7.4 with a solution having 8 grams oftris (hydroxymethyl) These tests indicate the influence of the alkylgroup on the efiicacy of the auxin ester in causing cell mortality. Thusit can be seen that the normal and the tertiary butyl esters ofind0lyl-3-acetic acid show a rate of kill four times greater than theethyl ester. The normal pentyl ester killed tumor cells about twice asrapidly as the ethyl ester of the indolyl-3-acetic acid. The methyl andnormal propyl esters were inferior to the ethyl ester in cytocidalactivity, but effected a mortality rate somewhat greater than observedin the controls.

EXAMPLE 3 The toxicity of indolyl-3-acetic acid and the esters thereofwas determined in a conventional manner by intraperitoneal injection ofthe test compound in cottonseed oil. These tests were conducted onWebster white Swiss mice. It was found that high dosages of tertiarybutyl ester, normal heptyl ester, normal octyl ester, normal nonyl esterand normal decyl ester could be tolerated by the animals. Somewhat lowerdosages were tolerable in the methyl ester, ethyl ester, propyl ester,isopropyl ester, normal butyl ester, normal pentyl ester and normalhexyl ester. The data for the 72 hour lethal dose (LD for certain auxinesters is presented in Table II.

aminomethane in ml. of water (tris butter) The mean survival time of anumber of animals were determined for both treated and untreated controlanimals.

The data for this test series wherein a semi-solid Ehrlich ascites tumorwas injected is set forth in Table III. The vehicle in this seriescomprised 18 rag/ml. 1,2 propanedi-ol, 3 mg./ml. Myvacet, and 5 mg./ml.Tween 20 made up to volume and bulfered as above. The auxin ester ofindolyl-3-acetic acid was suspended in said vehicle. The mean survivaltime is the numerical average of the days of survival of the testanimals, a plus sign indicating that the test series was terminatedbefore all of the animals had died and the balance were sacrificed.

TABLE III Number of Mean Surpentyl ester after transplant. vehicle.

It can be seen that the mean survival time of tumor infected animalshaving received a single treatment of the normal pentyl ester ofindolyl-S-acetic acid is at least 2 /2 times the mean survival time ofthe controls receiving only a saline injection.

EXAMPLE 5 TABLE IV Number of Mean Sur- Drug Drug Regimen Animals vivalTime 0.9% Saline 1 ml. daily for days- 19. 4 Vehicle 1 ml. on first day17 32. 3+

after transplant. 10 mgJml. tertiary 1 ml. 011 first day 20 40. 9+

butyl ester, 50 after transplant.

mg./ml. glucose in vehicle.

It can be seen that the treatment with the tertiary butyl ester ofindolyl-3-acetic acid more than doubles the mean survival time of miceinfected with a fatal carcinoma.

The data of Tables III and IV also show that an increase of meansurvival time is obtained by use of the vehicle alone. A nearlyidentical proportionate increase in mean survival time is indicated bythese data. It appears that the polysorbitan is the eificacious materialcausing this increase in survival and that it acts by increasing thepermeability of the cell wall so that the delicate balance of cellmetabolism is upset. The increase in survival time due to such a vehiclehas been observed for the semi-solid Ehrlich ascites carcinoma.

EXAMPLE 6 Another test series with a semi-solid Ehrlich ascitescarcinoma was conducted in a similar manner as Example 4. The vehicleemployed to suspend the auxin ester comprised mg./ml. 1,2 propanediol, 5mg./ml. Myvacet and 5 mg./ml. Tween 20 made up to volume with distilledwater and buffered as above. The data from this series are set forth inTable V.

TABLE V Number of Mean Sur- Drug Drug Regimen Animals vival Time 0.9%Saline 1 ml. on first day 15 15 after transplant. 10 mgJml. tertiary 1ml. on first day 20 40. 9+

butyl ester, after transplant. 50 mg./n1l. glucose in vehicle. 10 mg/ml.tertiary 1 ml. on first day 47. 3+

butyl ester, alter transplant +1 mgJml. glucose ml. daily of 50 mg./ invehicle. ml. glucose in vehicle. 10 mg./ml. tertiary 1 ml. on first day30 41. 2+

butyl ester, 3 mg./ alter transplant. ml. normal nndecyl ester, 50mg./ml. glucose in vehicle. 10 mgJml. normal 1 ml. on first day 30 45.5+

undecyl ester, 3 after transplant. mg./ml. tertiary butyl ester, 50 mg./ml. glucose in vehicle.

It can be seen from this series that all of the drug treatmentsincreased the mean survival time of the saline treated controls. It alsoappears that a beneficial effect is 8 obtained by administering glucosesolution to the test animals after auxin ester treatment. The injectionsof glucose made after the first day occur after the auxin ester isdegraded by the tumor cells so that it appears that no direct synergismis occurring.

EXAMPLE 7 A test series was conducted on a group of male Webster whiteSwiss mice injected with a liquid Ehrlich ascites carcinoma. This tumorwas transplanted by intraperitoneal injection as before. The treatmentinvolved intraperitoneal injection of drugs as described above. Thevehicle employed for the auxin ester comprised 25 mg./ml. 1,2propanediol, 5 mg./ml. Myvacet and 5 mg./ml. Tween 20 made up to volumewith 0.5% saline solution and buffered with tris buffer as above. Thedata from this series are set forth m Table VI.

TABLE VI Number of Mean Sur- Drug Drug Regimen Animals vival Time 0.9%Saline 1 ml. on first day 30 9. 3,

after transplant. Vehicle 1 ml. on first day 30 7. 2

after transplant. 1O fill/ml. tertiary 1 ml. on first day 30 12.2

butyl ester, 50 mg./ after transplant. ml. glucose in vehicle. 10mg./ml. tertiary 1 ml. on first day 30 10. 9

butyl ester, 3 mg./ after transplant. ml. undecyl ester, 50 mgJml.glucose in vehicle. 10 mg./ml. tertiary 1 ml. on first day 30 20. 9

butyl ester, 3 mg./ after transplant. ml. pentyl ester, 50 mat/ml.glucose in vehicle.

It can be seen from this series that some increase in mean survival timeis obtained in animals infected with a liquid Ehrlich ascites carcinomawhen treated with an auxin ester over the survival time of untreatedcontrol animals. The increases in survival time are greater thanstatistical variation in each instance, but not quite as great as theincreases when treating the less virulent semisolid tumor employed inExamples 4-6. The combination of tertiary butyl ester with the normalpentyl ester of indolyl-3-acetic acid doubles the lifetime of theanimals and demonstrates a synergism between these compounds.

EXAMPLE 8 A series of tests were performed with male Webster white Swissmice in the terminal stages of the Ehrlich ascites carcinoma. The miceemployed in these tests were implanted with the subject tumor aspreviously described seven days previous to the tests. Drugs wereinjected intraperitoneally in the mice as one milliliter of solutionseven days after transplant. The solution comprised the auxin ester in avehicle containing 18 mg./ml. 1,2 propanediol; 3 mg./ml. Myvacet; 5mg./ml. Tween 20, diluted and buffered to pH of 7.2 to' 7.4 with trisbuffer as recited above. One hour after injection of the test solutionthe animals were sacrificed and the ascitic fluid drained. A cell countwas made after staining the cells with Tyrodeeosin solution. Total freecell counts and stained free cell counts were made. The stained cellswere considered to represent dead cells. Control tests were madeemploying injections of glucose and injections of glutathione in thevehicle. A small percentage of dead cells was noted in both of thecontrols. Additionally doses of the normal pentyl and normal he-ptylesters of indolyl- 3-acetic acid were made either alone in the vehicleor in combination with glucose and glutathione in the vehicle. The dataobtained from this series of acute tests are set forth in Table VII.

TABLE vrr Drug Total cells Dead Cells Percent Kill Number AnimalsGlucose, 50 mgJml Glutathlone, 20 mg./ml

Normal pentyl ester, 25 mg./ml

Normal heptyl ester. 25 mg./ml

Glucose, 60 rngJml Glutathione, 2O mg./ml Normal pentyl ester, 20 mg./mlGlucose, 50 mg./ml Glutathione, 20 mg./ml Normal heptyl ester, 25 mgJmlt I I t ll l v It can be seen from these series of tests that asubstantial rate of kill of tumor cells in the ascitic fluid is obtainedby treatment with the normal pentyl and normal heptyl esters ofindolyl-3-acetic acid.

EXAMPLE 9 A group of 50 male Webster white Swiss mice were injected witha liquid Ehrlich ascites carcinoma as described above. On the first dayafter transplanting the tumor, each animal received a one milliliterdrug treatment. One half of the animals received 0.9% saline, and theother half received a suspension comprising 10 mg./ml. normal propylester of napthyl-l-acetic acid, 50 mg./ml. glucose, 25 mg./ml. 1,2propanediol, 5 rn-g./ml. Myvacet, and 5 mg./ml. Tween made up to volumeWith 0.5% saline solution and buttered with tris buffer as above. Thesaline treated animals showed a mean survival time of 9.5 days, whereasthose treated with the auxin ester showed a mean survival time of 24.3days, an increase of about 2% times.

Another group of 50 male Webster white Swiss mice received transplantsof a semi-solid Ehrlich ascites carminoma as described above. One-halfof this group received a saline treatment as above, and the other halfreceived treatment with the same suspension of normal propyl ester ofnpthyl-l-acetic acid as above. The saline treated animals showed a meansurvival time of 14.3 days, demonstrating that somewhat lower virulenceof the semi-solid reversion than the liquid tumor. The auxin estertreated animals showed a mean survival time of 21.6 days indicating asignificant increase in survival.

EXAMPLE 10 A group of 40 male Webster white Swiss mice receivedtransplants of a semi-solid Ehrlich ascites carcinoma by intraperitonealinjection. On the first day after transplant one half of the animalsreceived one milliliter of 0.9% saline solution. On the same day theother half of the animals received one milliliter of suspensioncomprising 20 mg./ ml. normal pentyl ester of napthyl-l-acetic acid, 50mg./ ml. glucose, mg./ml. 1,2 propanediol, 5 mg./ml. Myvacet, and 5mg./ml. Tween 20 made up to volume with 0.5% saline solution andbuffered with tris buffer as described above. The saline treated animalsshowed a mean survival time of 15.0 days, whereas the auxin estertreated animals showed a mean survival time of 56.1 days, an improvementsubstantially over 3 /2 times. Additionally the animals treated with theauxin ester no longer exhibited the semi-solid tumor transplanted, butrather were subject to a reverted hard tumor located in the peritonealcavity or elsewhere. Thus it is clear that the normal pentyl ester ofnapthyl-l-acetic acid caused a reversion of the Ehrlich ascitescarcinoma to a solid form.

It is to be understood that the above described examples are merelyillustrative of the application of the principles of this invention.Those skilled in the art may readily devise other variations that willembody the principles of this invention. It is therefore to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

What is claimed is:

1. A method for treatment of Ehrlich ascites carcinoma in micecomprising administering intraperitoneally in a mouse suffering withEhrlich ascites carcinoma an aqueous pharmaceutical carrier containingglucose and from 2 to 50rmilligrams of alkyl ester of an auxin permilliliter of carrier wherein said alkyl ester of an auxin is selectedfrom the class consisting of ethyl ester of indoly1-3-acetic acid,normal butyl ester of indolyl-3-acetic acid, tertiary butyl ester ofindolyl-3-acetic acid, normal pentyl ester of indolyl-3-acetic acid,normal heptyl ester of indolyl-3- acetic acid, and normal undecyl esterof indolyl-3-acetic acid.

2. A method of killing Ehrlich ascites carcinoma tumor cells in micecomprising administering intraperitoneally an aqueous pharmaceuticalcarrier containing glucose and from 200 to 600 milligrams of auxin esterper kilogram of body weight wherein said auxin ester is selected fromthe class consisting of ethyl ester of indolyl-3-acetic acid, normalbutyl ester of indolyl-3-acetic acid, tertiary butyl ester ofindolyl-3-acetic acid, normal pentyl ester of indolyl-3- acetic acid,normal heptyl ester of indolyl-B-acetic acid, and normal undecyl esterof indolyl-3-acetic acid.

3.. A method of killing Ehrlich ascites carcinoma cells in micecomprising intraperitoneally injecting a mouse with an aqueouspharmaceutical carrier containing an effective amount of an alkyl esterof an auxin for killing Ehrlich ascites carcinoma cells wherein saidalkyl ester of an auxin is selected from the class consisting of ethylester of indolyl-3-acetic acid, normal butyl ester of indolyl-3- aceticacid, tertiary butyl ester of indolyl-3-acetic acid, normal pentyl esterof indolyl-3-acetic acid, normal heptyl ester of indolyl-3-acetic acid,and normal undecyl ester of indolyl-3-acetic acid.

4. A method of killing Ehrlich ascites carcinoma cells in micecomprising intraperitoneally injecting a mouse with an aqueouspharmaceutical carrier containing glucose and an effective amount of analkyl ester of indolyl- B-acetic acid for killing Ehrlich ascitescarcinoma cells wherein the alkyl group is selected from the classconsist- 1 it ing of ethyl, normal butyl, tertiary butyl, normal pentyl,normal heptyl, and normal undecyl.

5. A method of killing Ehrlich ascites carcinoma cells comprisingintraperitoneally injecting a mouse with an aqueous pharmaceuticalcarrier containing glucose'and an effective amount of the ethyl ester ofindolyl-3-acetic acid for killing Ehrlich ascites carcinoma cells.

6. A method of killing Ehrlich ascites carcinoma cells comprisingintraperitoneally injecting a mouse With an aqueous pharmaceuticalcarrier containing glucose and an effective amount of the normal butylester of indolyl-3- acetic acid for killing Ehrlich ascites carcinomacells.

7. A method of killing Ehrlich ascites carcinoma cells comprisingintraperitoneally injecting a mouse with an aqueous pharmaceuticalcarrier containing glucose and an effective amount of the tertiary butylester of indolyl- 3-acetic acid for killing Ehrlich ascites carcinomacells.

8. A method of killing Ehrlich ascites carcinoma cells comprisingintraperitoneally injecting a mouse with an aqueous pharmaceuticalcarrier containing glucose and an effective amount of the normal pentylester of indolyl- 3-acetic acid for killing Ehrlich ascites carcinomacells.

9. A method of killing Ehrlich ascites carcinoma cells comprisingintraperitoneally injecting a mouse with an aqueous pharmaceuticalcarrier containing glucose and an effective amount of the normal heptylester of indolyl-3- acetic acid for killing Ehrlich ascites carcinomacells.

10. A method of killing Ehrlich ascites carcinoma cells comprisingintraperitoneally injecting a mouse With an aqueous pharmaceuticalcarrier containing glucose and an eifective amount of the normal undecylester of indolyl-3- acetic acid for killing Ehrlich ascites carcinomacells.

11. A chemotherapeutic composition comprising an aqueous solution ofglucose having therein a suspension of the normal butyl ester ofindolyl-3-acetic acid in a concentration of from 2 to 50 milligrams permilliliter of solution.

12. A chemotherapeutic composition comprising an aqueous solution ofglucose having therein a suspension of the tertiary butyl ester ofindolyl-3-acetic acid in a concentration of from 2 to 50 milligrams permilliliter of solution.

13. A chemotherapeutic composition comprising an aqueous solution ofglucose having therein a suspension of the normal pentyl ester ofindolyl-3-acetic acid in a concentration of from 2 to 50 milligrams permilliliter of solution.

14. A chemotherapeutic composition comprising an aqueous solution ofglucose having therein a suspension 12 of the normal heptyl ester ofindolyl-3-acetic acid in a concentration of from 2 to 50 milligrams permilliliter of solution.

15. A chemotherapeutic composition comprising an aqueous solution ofglucose having therein a suspension of the normal unde-cyl ester ofindolyl-3-acetic acid in a concentration of from 2 to 50 milligrams permilliliter of solution.

16. A chemotherapeutic composition comprising an aqueous solution ofglucose having therein a suspension of the tertiary butyl ester ofindolyl-3-acetic acid and the normal pentyl ester of indolyl-3-aceticacid with total concentration of said esters of from 2 to 50 milligramsper milliliter of solution.

17. A chemotherapeutic suspension comprising: a polysorbitan surfaceactive agent; acetylated monoglyceride; water; 1,2-propanediol; glucoseand an effective amount for chemotherapy of an alkyl ester ofindolyl-3-acetic acid wherein the alkyl group is selected from the classconsisting of ethyl, normal butyl, tertiary butyl, normal pentyl, normalheptyl and normal undecyl.

References Cited Chemical Abstracts (I) 51: 16836f (1957).

Chemical Abstracts (I) 51: Index page 1556s (under ethyl ester effect oncarcinoma) (1957).

Dyer, An Index of Tumor Chemotherapy, National Institute of Health,March 1949, pp. 10-12, 113 (No. 3185) and 21.

The Merck Index of Chemicals and Drugs, Seventh Edition, pp. 484, 485,490, 833, 863, and 970 (1960).

Aplfel et al., US. National Cancer Institute Journal, vol. 25, pp.ll11l120, October-December 1960.

Chemical Abstracts (III) 31: P4343 as indexed under 3-indoelaceticacid-alkyl esters on page 6589 of the Decennial Index, vols. 31-40, 1937to 1946.

Chemical Abstracts (II) 50: 17140b (1956) as indexed on page 8179 undermethyl ester-carcinogenic activity of Fifth Decennial Index M-OC.

Norman et al. (I), Nature, 198, p. 553, et seq. (1963).

Norman et al. (II), Nature, 199, p. 260, et seq. (1963).

Suguira, Cancer Research, vol. 25, No. 3, part 2, April 1965, pp. 494 to496 and 501 to 509.

ALBERT T. MEYERS, Primary Examiner.

SAM ROSEN, JULIAN S. LEVITT, Examiners. JEROME D. GOLDBERG, AssistantExaminer.

1. A METHOD FOR TREATMENT OF EHRLICH ASCITES CARCINOMA IN MICECOMPRISING ADMINISTERING INTRAPERITONEALLY IN A MOUSE SUFFERING WITHEHRLICH ASCITES CARCINOMA AN AQUEOUS PHARMACEUTICAL CARRIER CONTAININGGLUCOSE AND FROM 2 TO 50 MILLIGRAMS OF ALKYL ESTER OF AN AUXIN PERMILLILITER OF CARRIER WHEREIN SAID ALKYL ESTER OF AN AUXIN IS SELECTEDFROM THE CLASS CONSISTING OF ETHYL ESTER OF INDOLYL-3-ACETIC ACID,NORMAL BUTYL ESTER OF INDOLYL-3-ACETIC ACID, TERTIARY BUTYL ESTER OFINDOLYL-3-ACETIC ACID, NORMAL PENTYL ESTER OF INDOLYL-3-ACETIC ACID,NORMAL HEPTYL ESTER OF INDOLYL-3ACETIC ACID, AND NORMAL UNDECYL ESTER OFINDOLYL-3-ACETIC ACID.